Agr. Biol. Chem., 37 (12), 2813•`2819, 1973

_??_ties of Maltose Phosphorylase

from Lactobacillus brevis•õ

Atsumi KAMOGAWA, Kozi YOKOBAYASHI* and Toshio FUKUI

Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka, and *Hayashibara Co., Ltd., Okavama Received July 2, 1973

Maltose phosphorylase (EC 2.4.1.8) from Lactobacillus brevis was purified 29-fold over the crude extract. The final preparation was at least 80% pure and had a specific activity of 18 units/mg protein. The molecular weights of the native enzyme and of the component dissociated in sodium dodecyl sulfate were 150,000 and 80,000, respectively. The enzyme does not contain pyridoxal-5'-phosphate as a cofactor. It can not act on maltitol, malto triitol, sucrose, lactose and trehalose, and essentially not on isomaltose, maltobionic acid, maltotriose and maltotetraose. Inhibitory effect was observed with CuSO4, HgCl2 and p- chloromercuribenzoate. Some other properties were also examined. A possibility of using this enzyme for the analysis of maltose was proposed.

Maltose phosphorylase (maltose: orthophos MATERIALS AND METHODS phate glucosyltransferase, EC 2.4.1.8), dis Chemicals. Glucose, lactose, trehalose, sucrose, covered in Neisseria meningitidis by Fitting sodium arsentate and o-dianisidine•E2HCl were pur and Doudoroff1) and later in certain beer chased from Nakarai, Glucose oxidase (Aspergillus lactobacilli by Wood and Rainbow,2) catalyzes niger, Type II) and peroxidase (horseradish, Type II) the reversible phosphorolysis of maltose: were obtained from Sigma. Maltose (HHH), iso maltose, maltotriose, maltotetraose, maltitol, mal

Maltose + Pi = ƒÀ-glucose-l-P+ glucose totriitol and maltobionic acid were the products of Hayashibara Co., Ltd. To reduce the amount of

When arsenate is used in place of phosphate, contaminating maltose, these materials were pre treated with maltose phosphorylase for 90min at maltose yields two moles of glucose, and the 30•Ž, before use. Polypeptone and yeast extract were reaction proceeds to completion. Although the products of Daigo Eiyo. various monosaccharides were known to substitute for glucose to yield corresponding Determination ofglucose. Determination of glucose maltose analogues,1,3,4) the other properties liberated from maltose in the presence of arsenate was carried out according to the improved method of Lloyd of this enzyme have never been examined. We and Whelan.6) were interested in maltose phosphorylase for two reasons; if it is specific enough to be used Assay of maltose phosphorylase activity. Maltose in the determination of maltose in the presence phosphorylase activity was assayed by the determina tion of the glucose liberated from maltose in the pre of other maltooligosaccharides and if it con sence of arsenate. Standard assay system contained tains pyridoxal-5'-P which was found in all 0.2ml of 0.2M maltose, 0.2ml of 0.2M arsenate of the ƒ¿-glucan phosphorylases (EC 2.4.1.1) citrate buffer, pH 5.2, enzyme solution and water in a so far examined.5) This paper describes the total volume of 1ml. The reaction was started by the purification and properties of maltose phos addition of maltose. The mixture was incubated for 10min at 30•Ž and then boiled for 3min to stop the phorylase from Lactobacillus brevis. reaction. An aliquot of 0.1ml was removed and diluted to 1ml. After preincubation of the sample

and the glucose oxidase solution separately for 10min at 37•Ž, 2ml of the glucose oxidase solution were •õ Abbreviations: P, phosphate; P;, inorganic added to the sample and incubated for 30min at orthophosphate. 2814 A. KAMOGAWA, K. YOKOBAYASHI and T. FUKUI

37•Ž. The reaction was terminated by the addition of Wada and Snell.11)

4ml of 5N HCl, and the absorbance of the mixture

was read at 525nm. One unit of the enzyme activity RESULTS was defined as that amount of the enzyme which li

berates 2ƒÊmoles of glucose (corresponding to the Purification of maltose phosphorylase cleavage of 1 microequivalent of glucoside bond) per Preparation of crude extract. The cell min under the above conditions. The specific activity

was expressed by the enzyme activity per mg of pro paste (94g wet weight) which had been col tein.A lected from the 40 liter culture was mixed with

twice its weight of alumina and a small amount ssav of protein. Protein concentration was de of 5mM citrate buffer, pH 6.6, and ground in termined according to the method of Lowry et al.7) a mortar at room temperature for 90min. using bovine serum albumin as standard. The material was mixed with 280ml of the

Culture medium. The culture medium contained same buffer, and then centrifuged in the cold

peptone (1%), sodium acetate (1%), yeast extract at 9000•~g for 15min. The supernatant fluid (0.2%), MgSO4•E7H2O (0.02%), MnSO4•E4H2O thus obtained (240ml) was used as the crude (0.0002%) and maltose (1%). The pH was adjusted extract. to 7.0 with NaOH. The medium was sterilized by autoclaving for 15min at 120•Ž. Pro famine treatment and ammonium sulfate

Strain. Lactobacillus brevis IFO 3345 (ATCC fractionation. To 240ml of the crude ex tract was added 24ml of 2% protamine sul 8287) was used. fate. After stirring for 30min at 2•Ž, the Culture. One loopful of the cell suspension was solution was centrifuged at 9000•~g for 10min. inoculated in 10ml of the medium in a test tube and The supernatant fluid was then brought to incubated for 16hr at 27•Ž. The content was trans 0.4 saturation by the addition of solid am ferred to 1 liter of the medium and incubated for 8hr monium sulfate (54.5g) with stirring. After at 27•Ž without aeration. The culture was then

added to 20 liters of the medium and placed for 21hr 30min, the solution was centrifuged at 9000•~g at 27•Ž without aeration. The cells were harvested by for 10min, and the supernatant fluid was

centrifugation and washed once with 5mM citrate brought to 0.8 saturation by the addition of buffer, pH 6.6. solid ammonium sulfate (74g). After stirring for 30min, the solution was centrifuged at Disc electrophoresis. Polyacrylamide gel electro 9000•~g for 10min, and the supernatant solu phoresis was carried out according to the method of Ornstein and Davis8) with 7.5% gels in 0.05M Tris tion was discarded. The precipitate thus ob

glycine buffer, pH 8.9. The gels were stained for tained was dissolved in 30ml of 5mM citrate

protein with amido black. Polyacrylamide gel elec buffer, pH 6.6. trophoresis in sodium dodecyl sulfate was carried out by the procedure of Weber and Osborn9) with minor DEAE-cellulose chromatographies. All sub modifications. All gels were stained with coomassie sequent steps were carried out at 2•Ž. The brilliant blue. enzyme solution obtained in the above step Gel filtration. Chromatography on Sephadex was dialyzed against 4 liters of 5mM citrate

G-200 was carried out essentially as described by buffer, pH 6.6, for 20hr. The dialyzed solu Whitaker.10) Effluents in the chromatography were tion was applied to a column (3•~29cm) of monitored by absorption measurement at 230, 280 and DEAF-cellulose which had been equilibrated 340nm in a Hitachi effluent monitor. The void with 5mM citrate buffer, pH 6.6. Protein was volume was determined by using blue dextran. eluted with a linear gradient of zero to 1M Pyridoxal-5'-P content. A sample of the purified NaCl in 2 liters of 5mM citrate buffer, pH enzyme (4mg of protein) was mixed with perchloric 6.6. Fractions of 20ml each were collected acid to a final concentration of 0.3N of the acid. After and analyzed for protein and enzyme activity. centrifugation at 3000rpm for 15min, an aliquot of the supernatant solution was taken out and used for The fractions containing the enzyme activity the determination of pyridoxal-5'-P by the method of were pooled. The pooled enzyme solution Maltose Phosphorylase from Lactobacillus brevis 2815

FIG. 1. Hydroxylapatite Column Chromatography of Maltose Phosphorylase.

See text for the detailed procedure. •›•\•›, activity; •œ•\•œ, optical density at 280nm.

TABLE I. SUMMARY OF THE PURIFICATION OF MALTOSE PHOSPHORYLASE

FROM Lactobacillus brevisa)

a) From the 40-liter culture (94g wet weight cells).

was dialyzed against 4 liters of 5mM citrate buffer, pH 6.6, for 20hr. The dialyzed solu

tion was rechromatographed on DEAE- cellulose under the same conditions as above.

Hydroxvlapatite chromatography. The en zyme solution from the above step was dialyzed against three changes of each 5 liters of 1mm sodium phosphate buffer, pH 7.3. The dialyz ed solution was applied to a column (2.3•~

13cm) of hydroxylapatite (Hypertite C,

Clarkson) which had previously been equili brated with the same buffer. Protein was eluted with a linear gradient from 1mM sodium phosphate buffer, pH 7.3, to 0.2M FIG. 2. Disc Electrophoresis of the Purified Maltose sodium phosphate buffer, pH 7.1, in a total of Phosphorylase.

1 liter. Fractions of 10ml each were collect Electrophoresis was performed in 7.500 gel at pH 8.9, 4•Ž and 3mA/tube. The gel was stained with amido ed and analyzed for protein and enzyme ac black, and then scanned with a Fuji Riken densito tivity. Figure 1 shows a typical elution pattern meter FD-A IV using 600nm filter. 2816 A. KAMOGAWA, K. YOKOBAYASHI and T . FUKUI

of maltose phosphorylase from the hydroxylapatite columm. Fractions 13 to 22 were pooled and dialyzed overnight against

5 liters of 5mM citrate buffer, pH 6.6. The enzyme solution was concentrated with a collodion bag and stored at 2•Ž.

A summary of the purification is given in Table I. This procedure resulted in a 29- fold purification over the crude extract. The purity of the final preparation was more than 80% as judged by polyacrylamide gel electro phoresis (Fig. 2). Gel filtration on Sephadex G-200 gave a single elution pattern. This preparation was used throughout the follow ing experiments. FIG. 4. Stability of Maltose Phosphorylase against

pH. Stability Enzyme (80ƒÊg) was incubated in 0.5ml of the follow The purified enzyme preparation could be ing buffer at 3•Ž and given pH for 24hr. After ad stored either at -20•Ž or at 2•Ž for several justment to pH 5.4 with citrate or NaOH, a 50-ƒÊl months without appreciable loss of activity. aliquot was used for assay in the standard assay system. 50mM citrate buffer for pH 3.3•`6.4, 40mM Effect of heat on the stability of the enzyme is Tris-maleate buffer for pH 7.1, 50mM Tris-HCl buffer shown in Fig. 3. The enzyme was completely for pH 7.8, and 40mM glycine-NaOH buffer for pH stable at 35•Ž for 30min. However, the 8.7 and 9.7. activity was rapidly lost above 50•Ž. Figure Molecular weight 4 represents the pH stability of the enzyme. The molecular weight of native maltose Maltose phosphorylase was found to be stable over a range of pH 5.4 to 7.2. phosphorylase was determined by gel filtration on Sephadex G-200. The chromatography

gave a main symmetrical peak. Calculation

of the molecular weight by comparing its

elution volume divided by the void volume

(V/V0) with those of standard proteins yielded

FIG. 3. Stability of Maltose Phosphorylase against FIG. 5. Estimation of the Molecular Weight of Temperature. Maltose Phosphorylase by Sephadex G-200 Gel Filtration. Enzyme (20.8ƒÊg) was preincubated in 0.2ml of 20mM citrate buffer (pH 5.4) for 10 or 30min at 1, immunoglobulin G; 2, maltose phosphorylase; 3, various temperatures. Activity remained was assayed bovine serum albumin; 4, ovalbumin. V/V0, elution in the standard assay system. volume divided by the void volume. Maltose Phosphorylase from Lactobacillus br evis 2817

tially the same molecular size .

Absence of pyridoxal-5'-P Figure 7 shows the absorption spectrum of

maltose phosphorylase . It has a protein

absorption maximum at 280nm . No sign of the presence of pyridoxal-5'-P , which has an absorption in a region from 300 to 450nm , was obtained. The enzyme gave also negative results in the determination of pyridoxal or its 5'-P ester according to the phenylhydrazine FIG. 6. Estimation of the Subunit Molecular Weight of Maltose Phosphorylase by Sodium Dodecyl Sulfate method of Wada and Snell .11) It is thus con Disc Electrophoresis. cluded that maltose phosphorylase from Electrophoresis was carried out in 5% gel at 8mA Lactobacillus brevis does not contain pyridoxal- per tube. 1, immunoglobulin G; 2, muscle phos 5'-P as a cofactor. phorylase b; 3, maltose phosphorylase; 4, bovine serum albumin; 5, Taka amylase A; 6, alcohol dehy Effect of pH on the enzyme activity drogenase; 7, chymotrypsinogen; 8, myoglobin. Figure 8 shows the effect of pH on the

activity of maltose phosphorylase . The

enzyme is active over a range of pH 4.5•`7 .0 with the maximum activity around pH 5 .4 in arsenate-citrate buffer and in the 10-min incubation. Since the enzyme is unstable

below pH 5 (Fig. 4), the optimum pH might shift to a lower pH value when the activities

are determined in a shorter incubation time .

Km for substrates The reaction of maltose phosphorylase on

FIG.7. AbsorptionSpectrum of MaltosePhosphory lase. A, 0.46mgprotein/ml in 5mMcitrate buffer(pH 6.6); B, 5.5 mg protein/ml in the same buffer.

a value of 150,000 for maltose phosphorylase (Fig. 5). Upon polyacrylamide gel electro phoresis in sodium dodecyl sulfate the enzyme yielded a main band accompanied with several faint bands. The size of the main polypeptide chain was estimated by comparing its mobility with those of standard proteins (Fig. 6). The FIG. 8. Effect of pH on the Activity of Maltose molecular weight of polypeptide chain of Phosphorylase. maltose phosphorylase formed in the presence Reaction was carried out in the standard assay system of 1% sodium dodecyl sulfate was approximate except that the buffer was replaced by 40mM sodium ly 80,000. It appears that the enzyme is arsenate-citrate for pH below 5.6 and by 40mM sodium arsenate-HCl for pH above 5.6. Enzyme composed of two polypeptide chains of essen (20.8ƒÊg) was used. 2818 A. KAMOGAWA, K. YOKOBAYASHIand T. FUKUI various concentrations of substrates pro TABLE III. EFFECT OF METAL IONS AND SULFHYDRYL ceeded according to a typical Michaelis- REAGENTS ON THE ACTIVITY OF MALTOSE PHOSPHORYLASE Menten type reaction. Double reciprocal Reaction was carried out in the standard assay plots for the reaction velocities against the system in the presence of the given concentrations of substrate concentrations gave Km values of inhibitors. All inhibitor solutions were adjusted to

1.9, 1.7 and 2.6mM for maltose, arsenate pH 5.4. Enzyme (8.3ƒÊg) was used. and phosphate, respectively.

Substrate specificity Various oligosaccharides and their deriva tives were tested to see if maltose phosphoryla se can act on these materials liberating glucose. Since arsenate was used as the second sub strate, all of these materials should yield free glucose if the enzyme could act on them. The results are shown in Table II. Maltitol, maltotriitol, sucrose, lactose and trehalose were completely negative for the action of maltose phosphorylase. Slight action was a) Preincubated for 10min at 30•Ž . observed with isomaltose, maltobionic acid, maltotriose and maltoteraose, although the tion of maltose phosphorylase. No inhibi rate of glucose formation from these sub tory effect was also observed with isomaltose, strates was all less than 1% of that from maltitol, maltobionic acid, maltotriose, mal maltose. It is likely that the apparent actions totriitol, sucrose, lactose, trehalose (all 20mM) were caused by the contaminating maltose, and maltotetraose (10mM). The results with in spite of the pretreatment of the substrates metal ions and sulfhydryl reagents are shown with maltose phosphorylase. in Table III. All metal ions tested except Cu2+ and Hg` had no effect. Both mercury

Inhibitors compounds caused almost complete inhibition Glucose at the equimolecular concentration of the enzyme action at a relatively low con with maltose gave no effect on the rate of ac centration, although iodoacetamide was inert. Glucose-1-P, glucose-6-P, fructose-6-P, AMP TABLE II. SUBSTRATE SPECIFICITY OF and EDTA gave no effect. MALTOSE PHOSPHORYLASE

Reactions were carried out in the standard assay DISCUSSION system except for 90-min incubation. Enzyme

(20.8ƒÊg) was used. All the ƒ¿-glucan phosphorylases so far isolated from animals, plants and micro

organisms contain firmly bound pyridoxal-5'- P and are absolutely inactive without it.5)

Its direct role, however, is not clear and is a

subject of interest for many investigators. There has been no evidence whether pyri doxal-5'-P is present in other phosphorylases,

except sucrose phosphorylase which does not contain this cofactor.5) The present results

demonstrate that maltose phosphorylase from Lactobacillus brenis also does not contain Maltose Phosphorylase from Lactobacillus brevis 2819 pyridoxal-5'-P. REFERENCES Previous studies1,3,4) revealed that the 1) C. Fitting and M. Doudoroff, J. Biol. Chem., specificity of glucosyl acceptor in the reverse 199, 153 (1952). reaction of maltose phosphorylase is relatively 2) B. J. B. Wood and C. Rainbow, Biochem. J., broad; xylose, glucosamine, N-acetylglucos 78, 204 (1961). 3) Z. Selinger and M. Schramm, J. Biol. Chem., amine, and 2-deoxy-, 3-deoxy-, 6-deoxy-, 6- 236, 2183 (1961). deoxy-6-fluoro-, 2-O-methyl- and 6-O-methyl- 4) K. Morgan and W. J. Whelan, Nature, 196, 168 glucose can replace glucose, whereas mannose, (1962). 5) D. J. Graves and J. H. Wang, "The Enzymes," galactose, sorbitol and others are inert. The Vol. 7, 3rd ed., ed. by P. D. Boyer, Academic present experimental results show that maltose Press, Inc., New York and London, 1972, p. 435. phosphorylase can not act on maltitol, mal 6) J. B. Lloyd and W. J. Whelan, Anal. Biochem., totriitol, sucrose, lactose and trehalose, and 30, 467 (1969). essentially not on isomaltose, maltobionic 7) O. H. Lowry, N. J. Rosebrough, A. L. Farr and acid, maltotriose and maltotetraose. Thus, R. J. Randall, J. Biol. Chem., 193, 265(1951). 8) L. Ornstein and H. J. Davis, "Disc Electrophore the enzyme may be used as a tool for the sis," preprinted by Distillation Products Indus quantitative determination of maltose in the tries, Division of Eastman Kodak Co., Roches presence of starch hydrolysates. The method ter, New York, 1963. using this enzyme coupled with a glucose 9) K. Weber and M. Osborn, J. Biol. Chem., 244, oxidase system will be described in a separate 4406 (1969). 10) J. R. Whitaker, Anal. Chem., 35, 1950 (1963). paper.12) 11) H. Wada and E. E. Snell, J. Biol. Chem., 236, 2089 (1961). Acknowledgement. The authors are indebted to 12) A. Kamogawa, K. Yokobayashi and T. Fukui, Mr. S. Iwata for molecular weight determination. Anal. Biochem., in press.